Regulation of Steroid Hormone Production by Inter-Organelle Substrate Exchange

描述（由申请人提供）：类固醇激素（如皮质醇）是多种生物过程的关键调节剂，这些生物过程通过与转录因子的核受体超家族成员结合而引起生理变化。由于糖皮质激素控制许多基因的表达，因此使用多种机制来确保对激素产生的严格控制。我们的初步研究结果为促肾上腺皮质激素（ACTH）刺激的微管依赖性线粒体运输在维持最佳糖皮质激素输出中的作用提供了证据。我们还鉴定了几种蛋白质，包括GTdR RhoA和Rho效应子透明蛋白1（DIAPH 1），它们有助于这种运动。ACTH信号控制DIAPH 1与RhoA和其他几种结合伴侣相互作用的能力。然而，这些相互作用的功能意义尚未完全阐明，我们也没有定义，如果促肾上腺皮质激素刺激的线粒体运动，使皮质醇的生产，促进ER和线粒体之间的基板交付。我们推测ACTH刺激的线粒体运输速率的增加是细胞器间底物交换和糖皮质激素生物合成所必需的。为了验证这一假设，我们将定义ACTH信号控制DIAPH 1复合物组装的机制（具体目标1）。我们已经产生了一个稳定的细胞系，表达针对DIAPH 1的shRNA，以确定蛋白质在线粒体运动和激素产生中的作用。我们还将评估DIAPH 1和几种结合伴侣（包括微管蛋白、驱动蛋白和氧固醇结合蛋白相关蛋白2（ORP 2））之间相互作用的功能意义。还将进行研究以确定磷酸化在控制DIAPH 1功能中的作用（具体目标2）。我们已经发现ACTH信号刺激DIAPH 1的磷酸化。将使用磷酸化突变体和磷酸化特异性抗体来检查这种翻译后修饰对DIAPH 1功能的作用。最后，我们将确定11-脱氧皮质醇在ER和线粒体之间转运的机制（具体目标3）。我们发现ORP 2与DIAPH 1共纯化，并假设这种脂质结合蛋白可能促进ER和线粒体之间的底物转移。我们将描述ORP 2在皮质醇生物合成中的作用，并确定ER和线粒体之间的融合是否在细胞器间底物传递中发挥作用。从这些研究中获得的知识将提供有关肾上腺皮质类固醇激素合成的一个很大程度上未探索的方面的有价值的信息。我们的研究结果也可能提供洞察的机制，底物之间的ER和线粒体在其他代谢过程。

DESCRIPTION (provided by applicant): Steroid hormones such as cortisol are key regulators of a diverse array of biological processes that evoke physiological changes by binding to members of the nuclear receptor super family of transcription factors. Because glucocorticoids control the expression of numerous genes, multiple mechanisms are used to ensure tight control over hormone production. Our preliminary findings provide evidence for the role of adrenocorticotropin (ACTH)-stimulated, microtubule-dependent mitochondrial trafficking in maintaining optimal glucocorticoid output. We have also identified several proteins, including the GTPase RhoA and the Rho effector diaphanous 1 (DIAPH1), that act to facilitate this movement. ACTH signaling controls the ability of DIAPH1 to interact with RhoA and several other binding partners. However, the functional significance of these interactions has not fully been elucidated, nor have we defined if ACTH-stimulated mitochondrial movement enables cortisol production by facilitating the delivery of substrate between the ER and mitochondria. We hypothesize that ACTH-stimulated increases in the rate of mitochondrial trafficking are required for inter- organelle substrate exchange and glucocorticoid biosynthesis. To test this hypothesis we will define the mechanism by which ACTH signaling controls DIAPH1 complex assembly (Specific Aim 1). We have generated a stable cell line that expresses shRNA against DIAPH1 to define the role of the protein in mitochondrial movement and hormone production. We will also assess the functional significance of the interactions between DIAPH1 and several binding partners, including tubulin, kinesin, and the oxysterol- binding-protein related protein 2 (ORP2). Studies will also be performed to define the role of phosphorylation in controlling DIAPH1 function (Specific Aim 2). We have found that ACTH signaling stimulates the phosphorylaton of DIAPH1. The role of this post-translational modification on DIAPH1 function will be examined using phospho-mutants and a phospho-specific antibody. Finally, we will determine the mechanism by which 11-deoxycortisol is transported between the ER and mitochondria (Specific Aim 3). We have found that ORP2 co-purifies with DIAPH1 and hypothesize that this lipid binding protein may facilitate substrate transfer between the ER and mitochondria. We will characterize the role of ORP2 in cortisol biosynthesis and also determine if fusion between ER and mitochondria play a role in inter-organelle substrate delivery. The knowledge gained from these studies will provide valuable information about a largely unexplored facet of steroid hormone synthesis in the adrenal cortex. Our findings may also provide insight into the mechanism by which substrates are transferred between the ER and mitochondria in other metabolic processes.